GFP tagging of Ralstonia solanacearum facilitate its easy tracking in tomato cultivars without affecting virulence

Title : GFP tagging of Ralstonia solanacearum facilitate its easy tracking in tomato cultivars without affecting virulence

Abstract:

Green fluorescent protein (GFP) labelling of phytopathogenic bacteria Ralstonia solanacearum strain UTT-25 (Race 1, bv 3) has been used to study their infection, localization activity, movement at cellular level and monitoring bacterial disease development at whole-plant level in tomato resistant (Hawaii-7996) and susceptible (Pusa Ruby) cv. at different time intervals. This is a new method of isolation for direct-transformation of bacterial cells and easily introduced by electroporation, which was stably maintained with selective pressure (Kanamycin) in R. solanacearum (UTT-25) by using the binary vector pCambia1302 carrying gfp sequence as a reporter. After direct-transformation the cells were screened by selectable marker and further confirmed with PCR, fluorescence microscopy and Confocal Laser Scanning microscopy (CLSM). Important parameters which are critical for the quality of transformed cells of R. solanacearum and efficiency was standardized using green fluorescence protein assay, due to which we recorded the strong green fluorescence emitted from pCambia1302+UTT-25 cells. The infection in tomato plant tissues (leaf, stem and root) was done by using infiltration method. Accordingly, after 24hrs of infection it can be easily monitored using CLSM at wavelength (480-540nm), which targets live bacteria showing abundant green-fluorescing particles along the host cell periphery in CLSM, apparently in between the plasma membrane and the cell wall. These results suggested, pCambia1302 can serve as a direct easy-to-use GFP-tagging tool for any given strain of R. solanacearum in cytological as well as field studies. The use of GFP-labelled bacteria has a wide range of applications in host–bacterial interaction studies and bacterial ecology-related research.
Keywords: Ralstonia solanacearum, Tomato, pCambia1302, Fluorescence Microscopy, Confocal Laser Scanning microscopy (CLSM)

Biography:

Manju Sharma is professionally active since 1999 presently, working as Assistant Professor at Amity Institute of Biotechnology, Amity University Haryana, Gurgram, Delhi NCR, India  since May, 2013. Before, joining Amity, she served her two teaching contracts as Assistant Prof. with Jimma University, Department of Horticulture and Plant Sciences, College of Agriculture and Veterinary Medicine, Jimma, Ethiopia ,from 2009-2012 under UNDP programme. She earned her doctorate degree in Feb. 1999 In vitro improvement studies in Brassica  juncea L. and Sesamum indicum L.” at Department of Botany, University of Rajasthan, Jaipur, as  UGC project- JRF. She obtained her M.Sc. degree in Botany form the same department in 1989. She joined Department of Botany, Delhi University as UGC Project fellow from 1990-92.  She has actively participated & presented papers in many national & International conferences. She has published 17 research papers in international journals of repute, 5 books chapters and also wrote Book of poetry Wo jo rah gayee ankahee released in 5^th Delhi Literature Festival, New Delhi. Presently, she is guiding 5 Ph.D scholars. She also supervised exchange programme Ph. D work of RTF-DCS fellow on wheat resistance to Septoria from Addis Ababa University, Ethiopia from 29th Nov. 2015- 23rd May, 2016 funded by NAM-ST center, New Delhi. She has basic research background in plant tissue culture and gene transfer in important oil yielding crops. Presently, she is working on Expression of defense-related genes and signaling pathways in tomato, genetic relatedness and biochemical role of XopR-T3SS effector protein in Xanthomonas oryzae pv. Oryzae, molecular analysis of Phosphine resistance in Lesser grain borer Phyzopertha dominica infesting Wheat.